The composition and dynamic nature of the N-linked glycoprofile of bovine milk serum and its
individual proteins
Valk-Weeber, Rivca
DOI:10.33612/diss.134363958
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Valk-Weeber, R. (2020). The composition and dynamic nature of the N-linked glycoprofile of bovine milk serum and its individual proteins: A structural and functional analysis. University of Groningen.
https://doi.org/10.33612/diss.134363958
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7
Introduction
The evolutionary origin and possible
functional roles of FNIII domains in two
Microbacterium aurum B8.A granular
starch degrading enzymes, and in other
carbohydrate acting enzymes
Chapter 7
Quantitative analysis of bovine whey
glycoproteins using the overall N-linked whey
glycoprofile
Rivca L. Valk-Weeber
1, Talitha Eshuis-de Ruiter
2, Lubbert Dijkhuizen
1,3and
Sander S. van Leeuwen
1,41Microbial Physiology Research Group, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of
1Groningen, Groningen, The Netherlands
2Life Sciences, FrieslandCampina, Stationsplein 4, 3818 LE Amersfoort, The Netherlands 3Current address: CarbExplore Research BV, Zernikepark 12, 9747 AN Groningen, The Netherlands
4Current address: Laboratory Medicine, University Medical Center Groningen (UMCG), Hanzeplein 1, 9713 GZ, Groningen,
4The Netherlands
This work has been published in International Dairy Journal (2020) volume 110, article no. 104814
7
Abstract
Bovine whey is an important ingredient in human nutrition and contains many biofunctional, glycosylated proteins. Knowledge on the glycoprotein composition of whey and whey products is valuable for the dairy industry. This paper describes a method for the characterization of whey, or whey powders, by N-linked glycoprofile analysis. Application of the method for analysis of whey protein products showed clear differences in glycoprotein composition between concentrate, isolate and demineralized whey powders. The quantitative potential was explored by screening 100 pooled farm milk samples. IgG and lactoferrin protein concentrations determined by N-glycoprofile analysis matched well with ELISA results. The protein concentration of GlyCAM-1 was determined to be ≥ 1 mg/mL. The approaches presented in this work allow simultaneous concentration estimation of the three major whey glycoproteins, lactoferrin, IgG and GlyCAM-1 on the basis of their N-linked glycoprofiles, also in highly processed samples where conventional methods of detection (ELISA) are less suitable.
7
Introduction
Bovine milk is a complex mixture of water, lipids, carbohydrates (e.g. lactose), vitamins, minerals and proteins, which can be further divided into caseins and whey proteins (Korhonen, 2009). The whey proteins are the proteins present in the milk serum fraction, which remains after removal of the caseins. Acid whey is the fraction that remains in solution at 20°C when defatted milk is acidified to pH 4.6, resulting in disruption of the casein micelle structure and precipitation of casein proteins (Farrell et al., 2004). When making cheese via rennet treatment of milk, a sweet whey is produced that contains casein fragments (Thomä-Worringer et al., 2006).
Many whey proteins are bioactive glycoproteins occurring in varying quantities, such as lactoferrin (0.1-0.3 mg/mL), immunoglobulins G , A and M (IgG, IgA, IgM; 0.1-0.5 mg/mL), lactoperoxidase (0.03 mg/mL) and glycosylation dependent cellular adhesion molecule 1 (GlyCAM-1; PP3; lactophorin, 0.3-0.4 mg/mL) (Kussendrager & van Hooijdonk, 2000; Larson & Rolleri, 1955; O’Riordan et al., 2014b). The whey glycoproteins contain glycans of the N- or O-linked class defined by the amino acid side-chain to which they are linked. N-linked glycans can be further divided into three main classes, oligomannose, hybrid- or complex-type, depending on the monosaccharide composition of the glycan antennae present on the common core structure of two N-acetylglucosamine and three mannoses (GlcNAc2Man3)(Moremen et al., 2012). Some proteins, such as lactoferrin are exclusively N-glycosylated, while the casein fragment casein glycomacropeptide (GMP) is exclusively O-glycosylated (Moore et al., 1997; Vreeman et al., 1986). The protein GlyCAM-1 carries both N- and O-linked glycans (Kjeldsen et al., 2003). The N-linked glycans can be selectively released by peptide-N-glycosidase F. A chromatographic profile generated from the N-linked structures of whey (whey N-linked glycoprofile), is a complex mixture of structures (O’Riordan et al., 2014b; van Leeuwen et al., 2012b). Previously, we have shown that a glycoprofile obtained from acid whey is mostly the result of three main N-glycan contributing proteins: IgG, lactoferrin and GlyCAM-1 (Valk-Weeber et al., 2020b). Of these three main proteins, GlyCAM-1 contributes the highest total number of glycan structures to the whey N-glycan pool (Valk-Weeber et al., 2020b). The N-glycans of GlyCAM-1 are highly sialylated and fucosylated (Valk-Weeber et al., 2020b), both of which are important functional epitopes recognized as being involved in immune system modulation (Falconer et al., 2018; Johnson et al., 2013; Kaneko et al., 2006). The other main proteins, lactoferrin and IgG also have known antimicrobial and immune regulatory functions, mediated in part by their glycan structures (Figueroa-Lozano et al., 2018; Shields et al., 2002). Upon ingestion of milk and whey-based products, these proteins play an active role in the innate immune defense as well as acting as decoys for bacteria in the intestine (Li et al., 2019; Sauer et al., 2019; Ulfman et al., 2018).
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The components of whey, most notably the lactose and protein fractions, are commonly utilized in other products. Commercial whey powders, such as whey protein concentrates (WPC), and -isolates (WPI) are used as emulsifiers or texture enhancers (Fox & Mulvihill, 1982). Demineralized whey is often applied as a base ingredient of infant formula (Mettler, 1980). The protein composition and functional characteristics of these whey powders depends on the conditions used during their production, such as heat treatments or filtration steps (Morr & Ha, 1993; van Lieshout et al., 2019). Whey powders and the bioactive glycan structures contained therein are applied in various food products. The composition and glycoprofiles of proteins in bovine milk whey and whey powders are thus important to consider.
While multiple methods are available for the accurate determination of lactoferrin and IgG in milk or milk whey (Le et al., 2019; Palmano & Elgar, 2002), the options for GlyCAM-1 are much more limited. GlyCAM-1 can be determined by chromatography after separation from the other proteins (Paquet et al., 1988), but GlyCAM-1 often coelutes with other proteose proteins (Elgar et al., 2000; Innocente et al., 2011). Currently, no chromatographic technique is available for the determination of lactoferrin, IgG and GlyCAM-1 in a single analysis. In addition, regular protein chromatography does not generate additional information about the functional glycans. By analysis of the N-linked glycoprofile of (acid) whey proteins, or whey protein powders, the three main glycoproteins can be recognized in the glycoprofile. it was hypothesized that the glycoprofile allows protein concentration quantitation for lactoferrin, IgG and GlyCAM-1 by careful evaluation of key glycan structures in the obtained chromatograms. The concentrations of lactoferrin and IgG in milk are well established, yet the concentration of GlyCAM-1 is relatively unknown. By combining the quantitative and qualitative evaluation of single whey glycoprofiles, a large amount of data was generated concerning bovine whey glycoprotein composition and their glycan structures present.
Materials and methods
Materials
Reference bovine lactoferrin, and α-lactalbumin were provided by FrieslandCampina Domo (Amersfoort, the Netherlands). Bovine gamma globulin (purity > 98%) was from Serva (Heidelberg, Germany). For comparison, bovine IgG was isolated from mature milk using an 1 mL HiTrap Protein G HP antibody purification column (GE Healthcare, UK), according to manufacturer’s specifications. GlyCAM-1 was obtained via heat-treatment of milk as described by (Valk-Weeber et al., 2020b). PNGase F (Flavobacterium meningosepticum) was from New England Biolabs (Ipswich, UK). Aliquots of pooled tank milk
7
(quality control samples of pasture or regular milk), originating from 100 different individual farms in the Netherlands were obtained from FrieslandCampina Domo. Anonymized powders of whey protein concentrate (WPC), whey protein isolate (WPI) and demineralized whey products with different processing backgrounds were obtained from FrieslandCampina Domo.
Whey preparation and protein isolation
Samples were prepared according to the procedure of Valk-Weeber et al. (Valk-Weeber et al., 2020b). Milk samples were thawed in a water bath of 37 °C and homogenized. An aliquot of 1 mL was defatted by centrifugation at 4,000 x g for 10 min. An amount of 400 µL defatted milk was transferred into a new tube. Caseins were removed by addition of 125 mM of ammonium acetate at pH 4.6 at a ratio of 1:1. The samples were vortexed and left at room temperature for 5 min before centrifugation at 11,000 x g for 5 min to precipitate the caseins. An aliquot of 100 µL of the supernatant (acid whey) was transferred into a new tube and 400 µL 100 mM ammonium acetate in methanol (MeOH+NH4Ac) was added and mixed by vortexing. Whey protein precipitation was facilitated by centrifugation for 5 min at 11,000 x g. The solvent (containing lactose) was carefully pipetted from the protein pellets. The protein pellets were re-dissolved in 75 µL of 2% SDS and 2% β-mercaptoethanol in 80 mM phosphate buffer at pH 7.5. After addition of the solvent, the samples were incubated at 37 °C for 10 min, after which they were vortexed vigorously and further incubated for an additional 10 min, followed by a final vortex mixing. The proteins were denatured by heating for 15 min at 85 °C and cooled to room temperature. An aliquot of 25 µL of 10% NP-40 (NP-40 substitute, Sigma) was added to each sample and vortex-mixed. Finally, 2 µL of diluted PNGase F (100 units/experiment) was added to the samples and mixed. Glycans were released overnight at 37 °C.
Whey protein powder analysis
Samples of whey protein powders (WPC, WPI or demineralized whey) were dissolved in MilliQ water to give 20 mg/mL protein solutions, adjusted by their initial % protein (Table 1). An aliquot of 50 µL (corresponding to 1 mg of protein) was mixed with 50 µL of 125 mM of ammonium acetate solution at pH 4.6. Further processing including lactose removal by MeOH+NH4Ac and glycan release was performed as described above for the whey preparation method.
Labeling and cleanup
Isolated glycans were labeled with anthranilic acid (2-AA, Sigma) or 2-aminobenzamide (2-AB). The 2-AA label was chosen in combination with fluorescent detection in view of its higher sensitivity in these applications. The 2-AB label was chosen for applications that required mass spectrometry analysis. Direct in solution labeling of whey digests was performed as follows. Whey
7
protein digests of a total volume of 102 µL were mixed 1:1 with labeling solution (0.7 M 2-AA or 2-AB and 2 M of 2-picoline borane or sodium cyanoborohydride in dimethylsulfoxide (DMSO, Sigma): glacial acetic acid (7:3, v/v)). Incubations were performed for 2 h at 65 °C (Bigge et al., 1995). Labeling reagents were removed by 96-well microcrystalline cellulose SPE as described (Ruhaak et al., 2008). Samples were diluted with 612 µL acetonitrile (ACN) (final concentration 75% v/v) prior to application to the cellulose SPE.
Lactose determination
To quantitate lactose in supernatant and pellet after precipitation with MeOH containing 100 mM ammonium acetate, three milk samples were each processed in triplicate. In the final precipitation stage, the supernatant was carefully removed from the protein pellet and collected. The pellets were resuspended in 1 mL of MilliQ water. The supernatant fraction, containing MeOH, ammonium acetate and lactose, was evaporated under nitrogen flow, and the dry residue was resuspended in 1 mL of MilliQ. To verify the initial concentration of lactose in the selected samples, the defatted milk was centrifuged over a 10 kDa molecular weight cut-off filter (Amicon Ultra, Merck Millipore, Tullagreen, Cork, IRL) and collected. The concentration of lactose was determined against an 8-point calibration curve of lactose (lactose monohydrate, Sigma), from 2 µM to 2 mM. Samples were diluted to approximate 175 µM before analysis. High-Performance Anion-Exchange Chromatography (HPAEC) was performed on an ICS-3000 ion chromatography system (Thermo Fisher Scientific), equipped with a CarboPac PA-1 column (250 mm × 2 mm, Thermo Fisher Scientific). Detection was performed with a pulsed-amperometric detector (PAD) with a gold working electrode (four-step potential waveform) (Altunata et al., 1995). Lactose was eluted with a linear gradient of 10 mM NaOH + 2.5 mM sodium acetate to 40 mM NaOH + 25 mM NaOH in 25 min at a flow rate of 0.25 mL/min. The gradient was followed by a washing step with 100 mM NaOH and 600 mM sodium acetate for 5 min (0.25 mL/ min) and reconditioning to initial conditions.
Protein precipitation efficiency assay
In order to evaluate the efficiency of protein isolation via precipitation (see above), a comparison was made with protein recovery by desalting using molecular weight cutoff filters. The experiments were also performed on ultracentrifuged whey (100,000 x g, 1 h, 4°C, ~pH 6.7), prepared using a Sorvall RCM120GX micro-ultracentrifuge (Thermo Fisher Scientific, Waltham, MA). To test the effect of pH on the precipitation process, in addition to acid whey and ultracentrifuged whey, precipitation of neutralized acid whey (pH 7) was performed. Desalted whey was obtained by filtration of (acid or ultracentrifuged whey) using a molecular weight cutoff (MWCO) filter (10 kDa Amicon Ultra, Merck Millipore, Tullagreen, Cork, IRL). Protein precipitation of the different whey samples was carried out
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with MeOH+NH4Ac according to the whey preparation procedure as described above. The normally discarded supernatant (containing lactose) was collected to check for the presence of any protein. The supernatant was concentrated to approximately 20% of the original volume, by evaporation of the MeOH under a stream of nitrogen. A dilution was made to approximate 1 mL with MilliQ water and any protein present was recovered by centrifuging over a 10 kDa cutoff filter, as described above.
HPLC analysis
Fluorescently labeled glycans were separated on an Acquity UPLC Glycan BEH Amide column (2.1 mm x 100 mm, 1.7 µm, Waters Chromatography BV, Etten-Leur, the Netherlands), using an UltiMate 3000 SD HPLC system (Thermo Fisher Scientific, Waltham, MA) equipped with a Jasco FP-920 fluorescence detector (λex 330 nm, λem 420 nm, Jasco Inc, Easton, MD). An injection volume of 3 µL was used. Ternary gradients were run using MilliQ water, acetonitrile and a buffer solution consisting of 250 mM formic acid in MilliQ water, adjusted to pH 3.0 using ammonia. The buffer solution was maintained at a constant 20% throughout the run. Elution was performed by a slow sloping gradient of 22% to 40% MilliQ water (total concentration, including buffer) from 0 to 67.5 min. The remaining percentage of the solvent composition comprised of acetonitrile. Final gradient conditions were maintained for 9 min after completion of the gradient and the column reconditioned back to initial conditions for 13 min.
Determination of lactoferrin and Immunoglobulin concentration by ELISA
The concentrations of bovine immunoglobulins G, A and M and lactoferrin in the milk samples were quantified by the bovine ELISA quantitation kits (E10-118, E10-131, E10-101 and E10-126, respectively; Bethyl Laboratories, Montgomery, TX, USA). Manufacturer’s protocol were strictly followed, with the additional use of the Roche ELISA Blocking reagent as described below. ELISA plates (Costar, #3590) were coated with the capture antibody followed by a blocking step using Blocking reagent for ELISA from Roche (Roche Diagnostics, Mannheim, Germany, 11112589001). Milk samples were diluted within the calibration curve range (7.8 – 500 ng/mL for bovine IgG and lactoferrin and 15.6 – 1000 ng/mL for bovine IgA and IgM) using Blocking reagent for ELISA. As a last step, horseradish peroxidase (HRP)-labelled detection antibody was diluted in ELISA blocking reagent (Roche). TMB substrate solution (SB02, Thermo Fisher) was added to the wells. After 15 min, color development was stopped using 1 M HCl (Sigma). Absorbance was measured at 450 nm using a Bio-Rad iMark ELISA plate reader (Bio-Rad Laboratories, Hercules, CA, USA). Data were analyzed using a four-parameter logistic (4-PL) curve-fit.
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Lactoferrin calibration curve for glycoprofile-based determination of glyco-protein concentration
A calibration curve of lactoferrin was prepared in the range of 25 to 500 µg/mL in MilliQ. Samples for the calibration curve were diluted 1:1 with an 125 mM of ammo-nium acetate solution at pH 4.6; proteins in 100 µL aliquots were precipitated with 400 µL of MeOH+NH4Ac. For resuspension of protein pellets, glycan release, labeling and analysis, the whey preparation procedure was followed as described above.
Results and discussion
Sample preparation optimisation
To ensure efficient and repeatable analysis, the sample processing conditions were optimized. Acid whey contains glycoproteins of interest, as well as salts and a large quantity of lactose. To prevent competition between lactose and released glycans in the fluorescent labeling reaction, most lactose had to be removed from the samples. An effective approach for removal of lactose was a protein precipitation step of acid whey with 4:1 (v/v) MeOH containing 100 mM ammonium acetate (MeOH+NH4Ac). After removing MeOH+NH4Ac from the protein pellets obtained by centrifugation, analysis of the pellet and solvent fractions showed that lactose was > 95% reduced in the pellet fractions (Table S1). The protein precipitation of lactoferrin, IgG and GlyCAM-1 appeared complete with acidified (pH 4.6) whey, while a minimal loss occurred when using neutralized or ultracentrifuged (pH 6.7) whey (Fig. S1). This pH related precipitation efficiency of proteins, especially in the isoelectric point range of many proteins (pH 4-6), has been well described (Feist & Hummon, 2015). While sample loss appears minimal for neutralized or ultracentrifuged whey, it is recommended to acidify the whey (or whey powder solutions) to pH 4.6 prior to precipitation with MeOH+NH4Ac. Protein recovery was similar after precipitation or use of 10 kDa MWCO filters for desalting whey (Fig. S2). The obtained protein pellets were redissolved and digested by PNGase F, followed by direct in-solution reducing end labeling and subsequent cellulose cleanup in 96-wells plate format, adapted from a procedure described by Ruhaak et al. (2008). In the final protocol, 2-methylpyridine borane complex was preferred as catalyst over cyanoborohydride in view of its lower toxicity and lower sensitivity to water (Ruhaak et al., 2010b). Evaluation of both catalysts resulted in identical results under the optimized conditions (Fig. S3), fitting previous reports (Ruhaak et al., 2010b). With the optimized protocol, a sample set could be analyzed in just a few days, from whey preparation to overall glycoprofile analysis (Fig. S3).
Protocol repeatability
Ideally, an internal standard should be added to the raw milk samples to correct for any bias in the procedure. In view of the difference in degree of polymerization (DP) of an internal standard and the large DP range of the glycans obtained from whey, an added oligosaccharide is unlikely to be representative for the whole DP range and thus will not guarantee a good correction for all glycans present in the whey. In addition,
7
any added oligosaccharide added to the milk prior to sample processing, is lost during the protein precipitation step. Therefore, we opted to evaluate the method without use of any internal standard. The repeatability of the protein precipitation step was evaluated by a six-fold processing of three different samples of pooled milk. HPLC chromatograms of overall whey glycoprofiles were divided into 32 different complex-peak clusters and integrated (Fig. 1). The coefficient of variation (CV) was calculated for each peak cluster, over a total of 18 replicates between 3 samples. Variation was less than 6% for all integrated peak clusters (Table S2), indicating that the method was highly repeatable and sufficient for quantitative applications.
Figure 1. HPLC chromatogram of the overall bovine acid whey glycoprofile (A), divided into 32 separate
integrated peak clusters. The three main proteins that contribute N-glycans to the overall profile are lactoferrin, IgG and GlyCAM-1, as shown by the overlay of their individual glycoprofiles (B) at typical concentrations (GlyCAM-1 was analyzed directly from heated whey, lactoferrin 0.20 mg/mL, IgG 0.4 mg/mL). A complete N-glycan annotation of the whey glycoprofile was described earlier (Valk-Weeber, et al., 2020b). A selection of glycans from IgG (green asterisk), lactoferrin (pink asterisk) and GlyCAM-1 (blue asterisk) was annotated in the overlay chromatogram (B). The symbols used to represent the monosaccharides of a typical glycan structure are described in the legend. Mannose = Man, Galactose = Gal, N-Acetylneuraminic acid = Neu5Ac, Fucose = Fuc,
N-acetylgalactosamine = GalNAc, N-acetylglucosamine = GlcNAc. The oligomannose glycans
GlcNAc2Man5-9 are marked with the abbreviation Man-5 to Man-9. The three signature glycans of IgG, lactoferrin and GlyCAM-1, used for quantitation of their respective protein concentration, are
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Qualitative whey protein powder analysis
The main applications for the methods outlined here are glycoprofile analysis of acid whey prepared directly from milk. The methodology can be easily adapted for analysis of other sources of whey proteins. Instead of using acid whey, an aqueous protein solution can be mixed 1:1 with buffer at pH 4.6 to obtain a suitable solution for further processing. One such application is the analysis of commercial whey derived protein powders. The major whey protein powder products are demineralized whey, whey protein concentrate (WPC) and whey protein isolate (WPI) (Table 1). WPCs are obtained by processing the whey with ultrafiltration, ion exchange, or a combination thereof, reducing the lactose, water and mineral contents and increasing the protein content to 35-90% (Carter & Drake, 2018). WPIs are obtained by performing ultrafiltration after an initial microfiltration step. This microfiltration step removes lipids from the whey, and subsequent ultrafiltration yields a more pure and higher concentration WPI (Hanemaaijer, 1985). For infant formulas and products where a lower mineral content is preferred, demineralized whey is used, obtained by nanofiltration or ion-exchange (Jost et al., 1999; Mettler, 1980).
The glycoproteins present in whey determine its overall glycoprofile. The full whey glycoprofile contains a large number of N-glycans, which have been identified previously and attributed to lactoferrin, lactoperoxidase, α-lactalbumin, IgG and GlyCAM-1 (Valk-Weeber et al., 2020b). Of these five proteins lactoferrin, IgG and GlyCAM-1 contribute the majority of the structures found in the whey glycoprofile, as summarized in Fig. 1. Each protein contains unique signature glycans by which they can be identified in the overall profile (Valk-Weeber et al., 2020b) (Fig. 1). The glycoprofiles of a selection of commercial WPCs, WPIs and demineralized powders were analyzed and compared (Fig. 2). The WPC powders differed in their initial protein content (35 and 80%, respectively) (Table 1). Of each powder, a normalized amount of 1 mg of whey protein was analyzed. The glycoprofiles obtained for the 35% and 80% protein concentrates were generally very comparable, with some differences. Of the 35% WPC, the C1 glycoprofile displayed lower peak intensities than the rest (Fig. 2A). This is also the powder with the lowest (68%) Nitrogen Solubility Index (NSI), which is indicative of the soluble protein present in the whey powder. Since the powders were dissolved prior to analysis, any undissolved glycoprotein will not be taken into the analysis, resulting in a lower overall glycoprofile. Of the 80% WPC, the C10 and C13 (acid whey) glycoprofiles were most intense (Fig. 2B), indicating higher glycan (and thus glycoprotein) quantities.
The glycoprofiles of demineralized whey samples (Fig. 2C) were overall similar to those of WPC. Highest glycan peak intensities were observed for samples D2, D5, D6 and D7, while the overall lowest were observed for D3. In D3 the signature
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glycans of GlyCAM-1 were present in markedly lower intensity, indicating a lower concentration for this protein. The peak height of Man-9 (marked LF, Fig. 2C) in this powder, however, was similar to the other demineralized powders tested. This suggests that some glycoprotein (most notably GlyCAM-1) was lost during processing of this powder, but that lactoferrin was not affected.
Table 1. Whey powder types, processing methods, and protein content of the powders analyzed
in this study.a
Powder
Type Code WheyType Process Protein(%) NSI(%) Heat Treatment
Concentrate C1 Acid UF 35 68 Pasteurized
Concentrate C2 Cheese UF 35 83 Pasteurized
Concentrate C3 Cheese UF 35 93 Pasteurized
Concentrate C4 Cheese UF 35 87 Pasteurized
Concentrate C5 Cheese UF 35 93 Pasteurized
Concentrate C6 Cheese UF 80 N.A. Unknown
Concentrate C7 Cheese UF 80 N.A. Unknown
Concentrate C8 Cheese UF 80 N.A. Unknown
Concentrate C9 Cheese Unknown 80 N.A. Unknown
Concentrate C10 Acid UF 80 N.A. Pasteurized
Concentrate C11 Cheese UF 80 N.A. Pasteurized
Concentrate C12 Cheese UF 80 N.A. Unknown
Concentrate C13 Acid UF 80 N.A. Pasteurized
Isolate I1 Cheese UF+MF 90 N.A. Unknown
Isolate I2 Cheese Unknown 90 N.A. Unknown
Isolate I3 Cheese UF+MF 90 N.A. Unknown
Isolate I4 Cheese Unknown 90 N.A. Unknown
Isolate I5 Cheese Unknown 90 N.A. Unknown
Isolate I6 Unknown Unknown 90 N.A. Unknown
Isolate I7 Cheese Unknown 90 N.A. Unknown
Demineralized D1 Cheese Unknown 13 72 Pasteurized
Demineralized D2 Cheese Unknown 13 65 Pasteurized
Demineralized D3 Cheese IEX/NF/SF 13 82 Pasteurized
Demineralized D4 Cheese IEX/ED/NF 13 58 Pasteurized
Demineralized D5 Cheese IEX/NF 13 66 Pasteurized
Demineralized D6 Cheese ED/IEX 13 70 Pasteurized
Demineralized D7 Cheese ED/IEX 13 81 Pasteurized
Demineralized D8 Cheese ED/IEX/SF 13 74 Pasteurized
Demineralized D9 Cheese IEX 13 80 Pasteurized
aUsed abbreviations: Nitrogen Solubility Index (NSI), Ultra filtration (UF), micro filtration (MF), ion
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Figure 2. Overlays of the HPLC chromatograms obtained by glycoprofile analysis of WPC (A and B),
demineralized whey powders (C), and WPI (D). Glycoprofiles were determined for samples corresponding to 1 mg of protein. The peak clusters containing signature glycans (Fig. 1) of immunoglobulin G (IgG), lactoferrin (LF) and GlyCAM-1 (GCM) in the chromatogram are marked with (*). Peak clusters with the strongest Pearson correlation (Table 2) for the protein concentrations of IgG and LF are marked with (#).
WPC and demineralized whey thus appeared relatively similar in composition. Despite the highest protein content (90%), the glycoprofiles of WPI were markedly lower than the other two whey powders (Fig. 2D). The WPI glycans observed were primarily signature structures of GlyCAM-1. The WPI signature structures of IgG and lactoferrin were observed to have a much lower intensity compared to the other protein powders (Fig. 2). These proteins are thus likely to be removed from whey during the production process of WPI. The main difference between WPI and WPC production is the application of a microfiltration step for WPI production, suggesting that this step may result in glycoprotein loss. Qualitative evaluations of the commercial whey powder glycoprofiles, clearly demon-strated that differences in glycoprotein composition can occur due to processing conditions and protein concentration. For an accurate determination of glycoprotein concentrations, quantitation by integration of the obtained whey profiles was explored.
Peak cluster area and protein concentration correlation
Glycoprofiles of pooled bovine (tank) milk quality control samples from 100 individual farms in the Netherlands were analyzed and integrated according to Figure 1. Concentrations of lactoferrin, IgG, IgA and IgM were determined by ELISA (Table S1). Pearson correlations were made between the ELISA determined protein concentrations and the integrated glycan peak clusters (Table 2).
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Table 2. Correlations between ELISA determined protein concentrations and glycan peak clusters.Pearson correlations (r)a
Protein Peak clusterb LF IgG IgA IgM
2 IgG 0.56 0.57 ns 0.43 3 LF, GCM 0.78 0.48 0.55 0.56 4 LF, GCM, IgG 0.60 0.45 ns 0.42 6 LF, GCM, IgG 0.77 0.39 0.48 0.56 7 IgG, LF, GCM 0.70 0.47 0.50 ns 10 GCM, LF, IgG 0.49 0.55 ns 0.46 11 GCM ns ns ns ns 12 IgG, GCM, LF 0.59 0.78 0.53 0.59 13 LF, GCM 0.54 ns ns ns 16 GCM ns ns ns ns 17 GCM, LF 0.40 ns 0.47 ns 18 GCM, LF 0.62 ns ns ns 19 GCM ns 0.41 ns ns 21 LF, GCM, IgG 0.81 ns 0.48 ns 24 GCM ns ns ns ns 27 GCM. IgG 0.43 ns ns ns 28 GCM, IgG ns 0.42 ns ns 29 GCM, IgG 0.42 0.41 ns ns
aTwo-tailed Pearson correlations between protein concentration and peak cluster areas
(alpha = a0.01) were calculated in GraphPad Prism 7.0. Correlations were deemed significant when
a P avalue of < 0.0001 was obtained.
bPeak clusters integrated according to Figure 1. Peak clusters containing N-glycans of lactoferrin
b(LF), Immunoglobulin G (IgG) or GlyCAM-1 (GCM), according to (Fig. 1B) are marked with a bsuperscript (highest contributor first). Peak clusters without any significant correlation for any bof the tested parameters were excluded from the table. Abbreviations used: not significant (ns).
Several of the glycan peak clusters had a significant correlation with the ELISA determined protein concentration of lactoferrin, IgG, IgA and IgM (Table 2). The peak clusters showing significant correlations could be traced back to their protein of origin. For example, the oligomannose structures (GlcNAc2-Man5-9) of lactoferrin eluted under peak clusters 3 (Man-5), 6,7 (Man-6), 12,13 (Man-7), 17, 18 (Man-8) and 21 (Man-9), and peak cluster 4 containing a hybrid-type glycan of lactoferrin (Fig. 1; Table 2). Similar comparisons could be made for
7
IgG, with the most notable structure being a fucosylated di-antennary structure, GlcNAc2-Fuc-Man3-GlcNAc2-Gal2, eluting under peak cluster 12 (Fig 1.). A large number of glycan peak clusters (peak clusters 1, 5, 8, 9, 11, 14-16, 20, 22, 23, 25, 26, 30-32) did not correlate significantly with lactoferrin, IgG, IgA or IgM. These peak clusters have been associated with GlyCAM-1 (Fig. 1; Table 2) (Valk-Weeber et al., 2020b), a protein in whey of which the concentration has not been determined by ELISA.
The majority of the significant peak cluster to protein correlations found for lactoferrin and IgG ranged between r 0.40-0.60, yet for some clusters a higher correlation was found (r ≥ 0.70; Table 2). The intensity of peak clusters showing this higher correlation may to a large extent be determined by the eluting glycans of the corresponding glycoprotein. The strongest correlation (r = 0.81, P < 0.0001) was found for peak cluster 21 (Table 2), which corresponds to the Man-9 structure of lactoferrin (Fig. 1). This structure was selected for further exploration of lactoferrin quantitation by glycoprofile analysis.
Protein concentration determination: Lactoferrin
In previous work it was determined that the glycoprofile of lactoferrin in mature milk is highly constant over time and independent of inter-cow and breed differences (Valk-Weeber et al., 2020a). Of the total amount of glycans present on lactoferrin, 37.2% is the oligomannose Man-9 (Valk-Weeber et al., 2020a). Man-9 elutes in a relatively isolated section of the chromatogram (Fig. 1, peak 21), thereby making it a suitable target for integration and quantification.
A random selection of 12 individual samples was made out of the total of 100 pooled tank milk samples (Table S3). Quantification of the Man-9 peak was performed against a calibration curve prepared of a lactoferrin standard (Fig. 3). In order to estimate the concentration of the Man-9 peak, the whey glycoprofile peak area (Fig. 1, peak 21) had to be corrected for contributions by co-eluting structures from proteins other than lactoferrin (Fig. 1). Man-9 is also present on lactoperoxidase, however, its low protein concentration in combination with a relatively low percentage of Man-9 on this protein minimizes the contribution of lactoperoxidase to the Man-9 peak in the whey glycoprofile (Valk-Weeber et al., 2020b).
One of the co-eluting structures with Man-9 is a mono-sialylated structure from IgG (Fig. 1). The glycoprofiles of isolated IgG from milk in this study and commercial IgG from bovine blood serum were highly similar (Fig. S4). In addition, the IgG glycoprofile found in our study showed a high similarity with that in earlier work (Adamczyk et al., 2014; Fujii et al., 1990). It is thus likely that the IgG glycoprofile is conserved between cow populations. In this study, the use of pooled tank milk homogenized the IgG profile and the glycoprofile was assumed to be stable.
7
We analyzed the glycan distribution of IgG and calculated the percentages of its individual glycans (Fig. S5; Table S4). Using these percentages, by integration of a single IgG glycan, the area of the other glycan peaks in the profile can be calculated. To estimate the area contribution of co-eluting glycans of IgG with Man-9 (Fig. 1, peak 21), the area of the IgG signature glycan was integrated (Fig. 1, peak 2), and converted into the area of the interfering glycan under peak 21, allowing for an area correction.
Figure 3.
Flowchart for the determination of the concentration of lactoferrin, IgG and GlyCAM-1 by whey glycoprofile analysis. For lactoferrin, procedure A was followed, using peak cluster 21 (Fig. 1) and area correction for minor interfering glycans from IgG and GlyCAM-1 as calculated from peak 2 and 28, using the glycan distribution (Table S4 and S5, Fig. S5 and S6). For IgG and GlyCAM-1, concentration determination was performed by procedure B, calculating molar quantities of signature glycan peaks 2 and 28 (Fig. 1) and converting to the total glycan amount present per protein using their individual glycan distribution (Table S4 and S5, Fig. S5 and S6). The obtained molar quantities were converted to protein amounts by the N-glycan:protein ratio and molecular weight. The full procedure is detailed in the main text.
7
Finally, the co-eluting structures of GlyCAM-1 were considered, applying the same procedure as for IgG. The individual glycans of GlyCAM-1 were expressed as percentages of its total profile (Table S5 and Fig. S6). The signature glycan chosen for GlyCAM-1 was a tri-antennary glycan exclusive to GlyCAM-1 (Fig. 1, peak 28). The contribution to Man-9 (Fig. 1, peak 21), was estimated by calculating the co-eluting peak area of GlyCAM-1 at this position.
The average contributions of IgG and GlyCAM-1 to the total uncorrected concentration of lactoferrin were estimated as 0.02 mg/mL and 0.07 mg/mL, respectively (Table 3). The corrected lactoferrin concentration ranged from 0.14 to 0.28 mg/mL, which is within its normal reported range (Cheng et al., 2008). These values are, however, higher than our ELISA determined values (Table 3).
Table 3. Concentrations of lactoferrin, IgG and GlyCAM-1 determined in a selection of 12 pooled
milk samples by whey profile analysis and ELISA.a
Co-eluting contributionb
(mg/mL) Concentration(mg/mL)
IgG GlyCAM-1 Lactoferrin IgG GlyCAM-1
Methoda profile profile profile ELISA profile ELISA profile
Sample 1 0.02 0.08 0.24 0.15 0.43 0.36 1.41 2 0.02 0.08 0.21 0.18 0.43 0.50 1.42 3 0.02 0.06 0.28 0.21 0.39 0.38 1.09 4 0.02 0.06 0.21 0.16 0.48 0.45 1.13 5 0.01 0.07 0.19 0.14 0.36 0.44 1.22 6 0.01 0.06 0.15 0.11 0.37 0.37 1.10 7 0.01 0.07 0.25 0.17 0.26 0.40 1.23 8 0.02 0.07 0.25 0.17 0.39 0.39 1.23 9 0.02 0.06 0.19 0.11 0.43 0.40 1.09 10 0.02 0.06 0.20 0.16 0.40 0.41 1.01 11 0.01 0.07 0.14 0.10 0.38 0.38 1.30 12 0.01 0.07 0.16 0.10 0.35 0.33 1.17 Average 0.02 0.07 0.21 0.15 0.39 0.40 1.20
aQuantification of lactoferrin, IgG and GlyCAM-1 was performed by glycoprofile analysis (profile)
aaccording to Figure 3, or by ELISA. The following peak areas and structures were used for area acorrection; for IgG (Fig. 1: peak 2) for GlyCAM-1 (Fig. 1: peak 28).
bUsing the percentage distribution of the glycans for both IgG and GlyCAM-1 (Table S4; S5, Fig. S5; S6),
bthe area contribution of co-eluting structures with Man-9 of lactoferrin (Fig. 1: peak bcluster 21)
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The different methods used for lactoferrin detection may explain this discrepancy. The antibodies used for the ELISA detection of lactoferrin interact with conformational structures, which are lost upon denaturation of the lactoferrin protein (Van Berkel et al., 2002). Commercial ELISA kits for bovine lactoferrin determination are often used before and after treatments, in order to determine only the non-denatured fraction (Mazri et al., 2012; Teixeira et al., 2013). To quantify the full lactoferrin (native and denatured) pool, chromatographic tools are often used (Billakanti et al., 2019). Different methods (ELISA or chromatographic) are known to yield different concentrations of lactoferrin in the same sample (Indyk & Filonzi, 2005). finally, the ELISA detectable concentration of (human) lactoferrin is known to decrease significantly during storage; i.e. only 46% was detected after 6 months of frozen storage (Rollo et al., 2014). Considering that the bovine milk samples used in this study were stored frozen for > 6 months prior to lactoferrin determination by ELISA, a significant portion is likely denatured and thus remained undetected by ELISA. In contrast, lactoferrin quantitation by the glycoprofile analysis method operates independently of the folding of this protein. The method was not applied yet on fresh milk, which is an interesting target for future investigation of this hypothesis. For previously frozen milk, the protein precipitation and glycan extraction protocols described resulted in isolation of glycans from both folded and denatured protein molecules, allowing determination of the total lactoferrin concentration.
Protein concentration determination: IgG and GlyCAM-1
While lactoferrin is commercially available, this is not the case for GlyCAM-1. However, the concentrations of the IgG and GlyCAM-1 proteins in the whey can be estimated based on a calibration curve for lactoferrin (Fig. 3). For this purpose, the peak areas obtained were converted into molar quantities, using Man-9 of lactoferrin as a reference. Based on the carbohydrate content of lactoferrin (7.7%) (Valk-Weeber et al., 2019), and the Man-9 content (37.2%) (Valk-Weeber et al., 2020a), it was calculated that of the total carbohydrate weight, 2.87% originates from Man-9. For each gram of lactoferrin, 28.7 mg of Man-9 (1882.6 g/mol) is present, which corresponds to 15.2 nmol.
The glycans are fluorescently labeled at their reducing end; therefore, a single label is present on each glycan molecule. The detected fluorescent signal is a linear function of the number of fluorophores present. Using this information, a response ratio between the molar amounts of the detected fluorescent signal and the glycan structures present could be calculated. By interpolating the area of unknown peaks onto the calibration curve of the Man-9 peak, the molar amounts of the unknown peaks (and corresponding glycan structures) could be determined. This approach was used to calculate the molar amounts of the signature glycans of IgG (Fig. 1, peak 2) and GlyCAM-1 (Fig. 1, peak 28).
7
For IgG (Fig. 1, peak 2), this is a fucosylated di-antennary structure with a relative abundance of 10.7% (Table S4). For GlyCAM-1 (Fig. 1, peak 28), a tri-antennary structure was selected with a relative abundance of 5.5% (Table S5). Using the percentage of the individual signature glycans relative to the total amount of glycans present on a protein, the total molar amount of glycans for their respective proteins was calculated.
The amount of GlyCAM-1 protein has a direct 1:1 relation with its glycan quantity, since it contains a single 100% occupied N-glycosylation site (Sørensen & Petersen, 1993a). For IgG, the ratio of glycan to protein is 2:1, given the established 100% occupancy on both heavy chains (Takimori et al., 2011). In order to calculate the amount of protein, the molecular weight of the protein was used. For IgG, its molecular weight of 150 kDa is well established (Kickhöfen et al., 1968). For GlyCAM-1 however, the literature is inconsistent. On SDS-PAGE the PP3 (GlyCAM-1) fraction runs as a doublet of 28 kDa and 17 kDa., while the gene sequence codes for a protein with a mass of 15.3 kDa (Girardet & Linden, 1996; Sørensen & Petersen, 1993a). Its molecular weight including post-translational modifications by phosphorylation and both O- and N-glycosylation, has been determined as 19.4 kDa (Kjeldsen et al., 2003). For the protein concentrations calculated in this work, the molecular weight of 19.4 kDa was used.
The IgG and GlyCAM-1 concentrations determined by analysis of the whey glycoprofiles of 12 milk samples are displayed in Table 3. The concentrations for IgG ranged from 0.26-0.48 mg/mL when determined from the whey glycoprofile and 0.33-0.50 mg/mL when determined by ELISA. Highly similar results thus were obtained with both these methods; estimation of the IgG concentration from the whey glycoprofile seems a valid approach.
For GlyCAM-1 with a 19.4 kDa protein weight, concentrations determined by glycoprofile analysis ranged from 1.09-1.42 mg/mL. This GlyCAM-1 concentration range is significantly higher than its commonly accepted bovine milk concentration of 0.30-0.40 mg/mL (Bär et al., 2019; Larson et al., 1955). This lower concentration originates from analysis of heated milk, as a percentage of the total protein content, by proxy using protein nitrogen determination and electrophoretic separation of protein fractions (Larson & Rolleri, 1955). Larson & Rolleri stated that the determined concentration does not account for losses occurring during heating. Comparison of (un)heated whey glycoprofiles showed clear loss of GlyCAM-1 protein by heat treatment (Valk-Weeber et al., 2020b). Bär et al. recently quantified GlyCAM-1 by mass spectrometry analysis using reaction monitoring with isotopically labeled peptides and reported concentrations of 0.37 mg/mL in raw bovine milk and 4.2% wt g/100g) in whey isolated from raw milk. The concentrations of other proteins determined in the same study (bovine serum albumin, α-lactalbumin, lactoferrin) were far below (in the case
7
of BSA) or on the lower end of the concentration range commonly reported for these proteins. It is therefore possible that this method also underestimated the GlyCAM-1 concentration. These results show that GlyCAM-1 glycans dominate the glycoprofile of bovine whey, and that its protein concentration may in fact be much higher than previously reported.
Conclusions
Approaches for qualitative and quantitative characterization of whey, or whey powders, by N-linked glycoprofile analysis are presented. From a single analysis, the concentrations of the three main N-glycosylated proteins (lactoferrin, IgG and GlyCAM-1) can be estimated, as well as simultaneously generating data on the glycan structures present. The protocol is easily modifiable to facilitate analysis of protein powders, expanding the range to commercial whey products. The concentrations of IgG determined by this protocol were identical to its concentration determined by ELISA, while the lactoferrin concentration was slightly higher, but within the normal range reported for this protein. Particularly for (partially) denatured lactoferrin that cannot reliably be determined by ELISA, glycoprofile analysis may be a suitable proxy for its quantitation as it does not discriminate between native and denatured protein. Based on the concentrations of N-glycosylated proteins determined in this study, GlyCAM-1 has the highest concentration in bovine whey, followed by IgG and lactoferrin. This observation fits our previous result, showing that GlyCAM-1 glycans dominate the overall glycoprofile, despite its small size and single glycosylation site (Valk-Weeber et al., 2020b). The contrasting data regarding GlyCAM-1, both in terms of the protein properties, size and concentration, illustrates the need for further research towards this protein.
Acknowledgements
This work was financially supported by FrieslandCampina, the University of Groningen/Campus Fryslân, and the University of Groningen.
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Supplemental material
Table S1. Lactose concentrations in 3 diluted bovine whey samples, supernatants and pellets after
protein precipitation with 100 mM MeOH+NH4Ac (each analyzed in triplicate).a
Milk sample Whey (diluted) (µM) Supernatant (µM) Pellet (µM) 1 177 190 ± 6 5.6 ± 0.4 2 171 195 ± 3 6.2 ± 1.1 3 160 192 ± 9 6.3 ± 0.5
aWhey samples were diluted to an approximate 175 µM lactose concentration based on an
aestimated initial lactose concentration of +/-131 mM (calculated from a concentration of a45.1 mg/g milk) (Heck et al., 2009) and a density of 1.030 g/mL (Walstra et al., 2005).
Figure S1. Comparison of HPLC chromatograms of N-linked glycans obtained from whey proteins
recovered either by protein precipitation or molecular weight cutoff (MWCO) filters. Whey was either obtained by acidification to pH 4.6 (acid whey), or by ultracentrifugation. Protein precipitation with 100 mM ammonium acetate in methanol (MeOH) was carried out on the ultra-centrifuged or acid whey, or on neutralized acid whey. The proteins present in the methanolic fraction were recovered and the glycoprofile analyzed.
7
Table S2. Repeatability of the whey analysis method, expressed by the coefficient of variation (CV).aPeak cluster CV (%) Peak cluster CV (%) Peak cluster CV (%) Peak cluster CV (%) 1 LF, IgG 5.1 9 GCM 3.4 17 GCM, LF 3.3 25 GCM 5.1 2 IgG 5.0 10 GCM, LF 3.3 18 GCM, LF 3.1 26 GCM 4.9 3 LF 3.8 11 GCM 3.4 19 GCM 3.2 27 GCM 5.2 4 LF, GCM 3.3 12 IgG, GCM, LF 3.7 20 GCM 3.9 28 GCM 4.9 5 GCM, IgG 3.6 13 LF, GCM 3.7 21 LF 3.9 29 GCM 5.4 6 LF, GCM 4.6 14 GCM 3.0 22 GCM 6.1 30 GCM 5.5 7 IgG, LF 2.8 15 GCM 3.3 23 GCM 3.9 31 GCM 5.7 8 GCM, LF 3.5 16 GCM 2.5 24 GCM 5.5 32 GCM 5.4
aPeak clusters of 18 experiments (3 samples, 6 measurements per sample) were integrated
aaccording to Fig. 1 (main text). The CV was calculated for each peak cluster, over the 18 aexperiments. The main peak area contributors are marked with a superscript, Lactoferrin (LF), aImmunoglobulin G (IgG) and GlyCAM-1 (GCM).
Figure S2. HPLC chromatograms of N-linked glycans of whey proteins, after labeling with 2-AB
(top) and 2-AA (bottom) under different conditions. The catalyst used in the reaction was either sodium cyanoborohydride (NaCNBH3), or picoline borane. Labeling using picoline borane was also performed without the addition of acetic acid.
7
Table S3. Milk pr operties of 100 samples, ob tained fr om pooled milk fr om individual f arms. a Sample IgG IgA IgM LF Fa t Pr ot ein Lact ose to ta l Sample IgG IgA IgM LF Fa t Pr ot ein Lact ose to ta l µg /mL µg/mL µg /mL µg /mL w/v% w/v % w/v % Lit er s µg /mL µg /mL µg /mL µg /mL w/v % w/v% w/v % Lit er s 1 395 46 51 120 4.66 3.50 4.56 29384 51 503 60 98 222 5.41 4.05 4.51 3395 2 (1) 355 43 52 149 4.18 3.60 4.67 9922 52 (12) 509 61 69 232 4.26 3.66 4.55 12006 3 515 55 84 151 4.88 3.72 4.54 2653 53 428 47 65 200 4.58 3.71 4.55 6348 4 336 31 42 100 4.57 3.44 4.63 7802 54 466 40 56 125 4.71 3.58 4.54 9562 5 (2) 502 48 62 179 4.69 3.75 4.55 5405 55 334 39 39 102 4.81 3.83 4.72 716 6 498 55 67 175 4.64 3.52 4.61 9395 56 375 41 48 97 4.45 3.52 4.54 10687 7 486 45 69 153 4.85 3.80 4.48 9148 57 345 32 37 98 4.11 3.39 4.56 10656 8 467 40 59 212 4.67 3.84 4.55 6460 58 557 44 66 162 4.71 3.67 4.52 5315 9 493 43 55 134 4.55 3.70 4.56 4800 59 393 47 61 141 5.37 3.9 4.4 5663 10 (3) 375 53 74 213 4.78 3.99 4.53 6141 60 524 63 78 247 4.69 3.91 4.54 6727 11 507 59 80 191 5.05 3.76 4.51 6205 61 420 N.A . N.A . N.A . 4.97 3.64 4.53 2400 12 554 53 72 174 4.28 3.49 4.54 5824 62 477 N.A . N.A . N.A . 4.67 3.61 4.57 5970 13 449 67 77 197 5.14 3.92 4.52 6179 63 386 N.A . N.A . N.A . 4.53 3.59 4.52 9908 14 492 45 62 170 4.85 3.78 4.64 2609 64 451 N.A . N.A . N.A . 4.51 3.72 4.62 13015 15 (4) 450 64 77 163 4.97 3.75 4.51 4643 65 423 N.A . N.A . N.A . 4.00 3.59 4.56 10023 16 399 51 72 135 4.79 3.74 4.51 6963 66 521 N.A . N.A . N.A . 4.59 3.57 4.5 1173 17 366 39 57 105 4.74 3.56 4.53 6667 67 423 N.A . N.A . N.A . 4.71 3.69 4.57 8048 18 463 41 54 145 4.84 3.51 4.47 5441 68 406 N.A . N.A . N.A . 4.46 3.72 4.54 4627 19 403 42 46 147 4.29 3.62 4.58 21762 69 418 N.A . N.A . N.A . 4.93 3.66 4.52 4952 20 (5) 442 40 61 136 4.59 3.68 4.63 5110 70 495 N.A . N.A . N.A . 4.87 3.66 4.57 20130 21 643 67 72 242 4.94 3.71 4.45 2420 71 596 N.A . N.A . N.A . 4.95 3.83 4.47 4498 22 439 53 73 149 4.65 3.64 4.53 2083 72 507 N.A . N.A . N.A . 4.39 3.48 4.53 8829 23 577 41 100 131 4.13 3.34 4.61 416 73 492 N.A . N.A . N.A . 4.70 3.6 4.42 1020 24 370 52 66 156 4.59 3.62 4.59 6589 74 432 N.A . N.A . N.A . 4.80 3.62 4.6 5489 25 (6) 370 32 45 106 4.75 3.49 4.55 5203 75 438 N.A . N.A . N.A . 4.45 3.44 4.49 61977
Sample IgG IgA IgM LF Fa t Pr ot ein Lact ose to ta l Sample IgG IgA IgM LF Fa t Pr ot ein Lact ose to ta l µg /mL µg/mL µg /mL µg /mL w/v% w/v % w/v % Lit er s µg /mL µg /mL µg /mL µg /mL w/v % w/v% w/v % Lit er s 26 568 44 68 109 4.64 3.48 4.57 4033 76 528 N.A . N.A . N.A . 4.99 3.9 4.55 2407 27 639 71 100 225 4.86 3.92 4.45 8257 77 526 N.A . N.A . N.A . 4.78 3.55 4.56 3662 28 430 44 60 141 4.57 3.66 4.59 6578 78 532 N.A . N.A . N.A . 4.48 3.48 4.55 2954 29 551 54 86 140 5.47 3.95 4.51 805 79 454 N.A . N.A . N.A . 4.76 3.75 4.52 3313 30 (7) 397 37 62 166 4.64 3.61 4.51 3415 80 456 N.A . N.A . N.A . 4.71 3.84 4.56 7250 31 445 36 49 115 4.58 3.67 4.58 11374 81 421 50 64 211 4.60 3.73 4.57 13846 32 325 30 35 72 4.04 3.41 4.62 9418 82 383 32 49 148 4.77 3.58 4.47 3974 33 445 38 52 146 4.54 3.66 4.56 13394 83 456 47 88 213 5.32 3.97 4.52 1687 34 475 48 65 128 5.01 3.60 4.46 3533 84 432 59 64 177 4.65 3.77 4.54 9837 35 (8) 390 51 54 174 4.7 3.75 4.56 11489 85 555 56 72 205 5.24 3.73 4.4 3120 36 477 52 62 151 4.58 3.69 4.49 5211 86 469 48 68 175 4.65 3.77 4.56 16437 37 362 36 54 131 4.56 3.76 4.53 9051 87 424 42 64 176 4.82 3.83 4.57 3804 38 386 35 59 113 4.64 3.60 4.53 2981 88 351 26 39 85 4.30 3.55 4.59 3913 39 501 51 60 153 4.63 3.71 4.51 20458 89 356 35 51 127 4.90 3.68 4.52 5900 40 (9) 395 45 60 106 4.69 3.71 4.61 7501 90 467 29 49 101 4.59 3.78 4.56 6169 41 473 47 55 127 4.23 3.53 4.6 5397 91 562 51 76 201 5.10 3.92 4.36 1439 42 401 40 52 105 4.19 3.60 4.64 6240 92 359 31 50 127 4.72 3.55 4.46 2504 43 438 42 53 124 4.38 3.65 4.6 3488 93 344 33 55 115 4.54 3.56 4.55 5984 44 426 48 59 142 4.83 3.78 4.53 6787 94 412 52 67 177 4.44 3.54 4.47 3472 45 (10) 405 41 46 162 4.75 3.59 4.52 6104 95 501 46 62 175 4.81 3.81 4.55 8370 46 444 56 61 186 4.66 3.74 4.54 12243 96 488 43 64 176 4.58 3.72 4.53 5373 47 480 58 71 197 4.44 3.79 4.55 19074 97 622 54 74 140 4.72 3.83 4.64 1801 48 453 36 60 132 4.74 3.76 4.49 4647 98 456 40 56 156 4.80 3.76 4.49 3831 49 430 42 61 169 5.1 3.84 4.43 3130 99 351 34 45 151 4.68 3.77 4.56 6279 50 (11) 376 44 56 99 4.19 3.54 4.64 20149 100 418 37 52 143 4.41 3.52 4.5 5917 The tot al amoun t of tank milk for each sample is giv en as “t ot al lit er s” . Concen tra tions of IgG, IgA , IgM and LF w er e de termined by ELIS A. Samp les select ed for LF, IgG and GlyCAM-1 c
oncen tra tion de termina tion b y gly copr ofile analy sis ar e br ack et ed with number s 1-12.
7
Figure S3.
Flow diagram of the final protocol for N-glycan isolation and analysis in bovine milk samples, including an indication of the time required for each step.
Figure S4. Comparison of HPLC chromatograms obtained for the glycoprofiles of commercial
(bovine serum) IgG, and IgG isolated from milk in this study. * indicates the peak used for normalization of chromatograms.
HPLC chromatogram and integrated peaks of the glycoprofile of bovine serum IgG; for structural assignments, see Table S4.
7
Table S4. Percentage glycan distribution of individual glycan structures of IgG.Peaka Structuresb % of total Molecular mass
Underivatized monoisotopic 1 2 2.4 ± 1.2 1316.4865 2 * 6 10.7 ± 1.5 1462.5444 3 8 5.3 ± 0.9 1478.5393 4 12 7.7 ± 0.3 1624.5973 5 12 23.9 ± 0.7 1624.5973 6 25 2.7 ± 0.5 1827.6766 7 14 2.4 ± 0.4 1640.5922 8 23 23.5 ± 0.7 1786.6501 9 34 2.3 ± 0.1 1989.7295 10 30 2.7 ± 0.7 1931.6876 11 32 3.0 ± 0.6 1947.6825 12 # 42 4.5 ± 0.2 2093.7404 13 48 1.6 ± 0.2 2222.7830 14 50 1.9 ± 0.3 2238.7779 15 57 2.0 ± 0.6 2254.7728 16 51 57 3.2 ± 1.0 2400.8307 (1)2254.7728 (2) 2327.8018 (avg) 17 57 1.2 ± 0.5 2254.7728 18 65 0.4 ± 0.1 2561.8632 Total 100%
aPeaks according to Supplementary material Fig. S5, Structure selected for quantification, marked
a*; structure co-elutes with Man-9 of lactoferrin, marked #. An underivatized molecular mass (Da) a of the glycan structures is given. An average (avg) mass of an individual glycan on IgG is provided, a calculated by combining the percentage contribution with the underivatized glycan mass. When a multiple structures were appointed to a single peak, all were assumed to contribute equally and a an average was calculated.
7
Table S5. Percentage glycan distribution of individual glycan structures of heated whey (GlyCAM-1).
peaka Structuresb % of total Molecular mass Underivatized monoisotopic
peaka Structuresb % of total Molecular mass Underivatized monoisotopic 1 2 1.3 ± 0.3 1316.4865 18 47 0.9 ± 0.0 2192.8088 2 1 0.6 ± 0.0 1234.4334 19 40 9.0 ± 0.0 2077.7455 3 7 0.7 ± 0.0 1478.5393 20 38 45 2.5 ± 0.0 2036.7189 (1) 2151.7823 (2) 2094.2506 (avg) 4 19 2.2 ±0.0 1722.6453 21 56 0.2 ± 0.0 2378.8729 5 13 17 14 2.2 ± 0.0 1624.5973 (1) 1681.6187 (2) 1640.5922 (3) 1648.9361 (avg) 22# 56 54 1.7 ± 0.0 2378.8729 (1) 2337.8463 (2) 2358.3596 (avg) 6 26 4.9 ± 0.0 1868.7032 23 54 1.4 ± 0.1 2337.8463 7 15 0.8 ± 0.0 1640.5922 24 62 54 2.6 ± 0.1 2524.9308 (1) 2337.8463 (2) 2431.3886 (avg) 8 25 11 22 4.5 ± 0.1 1827.6766 (1) 1599.5656 (2) 1786.6501 (3) 1737.9641 (avg) 25 62 3.3 ± 0.1 2524.9308 9 23 1.6 ± 0.0 1786.6501 26 53 3.5 ± 0.1 2296.8198 10 37 2.4 ± 0.1 2013.7407 27 60 4.8 ± 0.0 2483.9042 11 20 0.7 ± 0.0 1728.6082 28 50 55 2.4 ± 0.1 2238.7779 (1) 2368.8409 (2) 2303.8094 (avg) 12 29 1.7 ± 0.1 1915.6927 (1) 1972.7141 (2) 1944.2034 (avg) 29* 59 5.5 ± 0.1 2442.8777 13 34 3.7 ± 0.0 1972.7141 30 51 0.6 ± 0.0 2254.7728 14 33 6.3 ± 0.1 2159.7986 31 63 67 1.4 ± 0.0 2529.8733 (1) 2702.9785 (2) 2616.4529 (avg) 15 20 4.1 ± 0.0 1931.6876 32 57 66 64 3.0 ± 0.0 2400.8307 (1) 2661.9520 (2) 2545.8683 (3) 2536.2170 (avg) 16 44 4.5 ± 0.0 2118.7720 33 68 3.0 ± 0.2 2808.0099 17 43 11.9 ± 0.1 2118.7720 Total 100%
aPeaks according to Supplementary material Fig. S6. Structure selected for quantification, marked
a*; Structure co-eluting with Man-9 of lactoferrin, marked #. An underivatized molecular mass of athe glycan structure is given.
bStructures were numbered according to the full profile annotation, as determined by Valk-Weeber
